Derivation of embryonic stem cells from cloned blastocysts using improved somatic cell nuclear transfer in common marmosets

The common marmoset (Callithrix jacchus) is a genetically modifiable non-human primate increasingly used in biomedical research. Here, we established a method for deriving embryonic stem cells (ESCs) from blastocysts generated by somatic cell nuclear transfer (SCNT) in the marmoset. Injection of histone demethylase Kdm4d mRNA enabled efficient reprogramming of somatic nuclei, allowing blastocyst formation in 14.5% from fibroblasts. Combining this method with a G9a/EHMT2 histone methyltransferase inhibitor improved blastocyst quality and allowed derivation of nuclear transfer ESCs (ntESCs), including wild-type and GFP-transgenic lines. These ntESCs exhibited normal karyotypes and pluripotency. Nuclear and mitochondrial DNA analyses confirmed their nuclear donor origin and cytoplasmic inheritance from recipient oocytes. Transcriptome analysis identified abnormally expressed genes in ntESCs present in a line-dependent and independent manner, suggesting partial reprogramming resistance. Our study establishes a marmoset SCNT method enabling derivation of ntESCs and provides a new platform for preserving and engineering marmoset genetic resources.